Establishment of a Cultivated Human Conjunctival
Epithelium as an Alternative Tissue Source for
Autologous Corneal Epithelial Transplantation
Hidetoshi Tanioka,1 Satoshi Kawasaki,1 Kenta Yamasaki,1 Leonard P. K. Ang,1,2
Noriko Koizumi,2 Takahiro Nakamura,1 Norihiko Yokoi,1 Aoi Komuro,1
Tsutomu Inatomi,1 and Shigeru Kinoshita1
PURPOSE. The corneal epithelium is essential for maintaining
corneal transparency, and efforts have been made to develop
improved techniques for corneal epithelial transplantation in
patients with total limbal failure. We evaluated the suitability of
transplanted cultivated human conjunctival epithelium (HCjE)
as a corneal epithelium replacement in rabbits with total cor-
neal and limbal deficiency.
METHODS. HCjE cells, cultivated on human amniotic membrane
(AM) to confluence and exposed to an airliquid interface
(air-lifted), were transplanted onto denuded rabbit corneas and
monitored for 2 weeks. The cultivated HCjE sheet and the
engrafted epithelium were analyzed by immunohistochemistry
and transmission electron microscopy (TEM).
RESULTS. The transplanted HCjE remained transparent, smooth,
and without epithelial defects during the follow-up period.
Both the cultivated HCjE cells and the engrafted epithelium
manifested five to six layers of stratified squamous epithelium
similar in morphology to normal corneal epithelium. The basal
cells expressed the putative stem cell markers (ABCG2 and
P63) and hemidesmosome and desmosome component pro-
teins. The cytokeratins (CK4, CK13, CK3, and CK12) and
MUC4 were found in the engrafted epithelium. However,
MUC5AC was not expressed. The results indicate that HCjE
cultivated on AM has the potential to be used as an alternative
corneal epithelium.
CONCLUSIONS. The transplantation of cultivated HCjE sheets is a
promising technique for the treatment of eyes with limbal
failure. (Invest Ophthalmol Vis Sci. 2006;47:3820 3827) DOI:
10.1167/iovs.06-0293
T he ocular surface is covered by at least two different types
of epithelia: corneal and conjunctival.1 4 These two epi-
thelial tissues are indispensable in keeping homeostasis of the
eye by expressing various specific genes such as cytokeratin
From the 1Department of Ophthalmology, Kyoto Prefectural Uni-
versity of Medicine, Kyoto, Japan; and the 2Singapore National Eye
Center, Singapore.
Supported by Grant-in-Aid 16390502 for scientific research from
the Ministry of Education, Science, Culture, and Sports of Japan.
Submitted for publication March 18, 2006; revised April 27, 2006;
accepted July 14, 2006.
Disclosure: H. Tanioka, None; S. Kawasaki, None; K. Ya-
masaki, None; L.P.K. Ang, None; N. Koizumi, None; T. Nakamura,
None; N. Yokoi, None; A. Komuro, None; T. Inatomi, None; S.
Kinoshita, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked advertise-
ment in accordance with 18 U.S.C. 1734 solely to indicate this fact.
Corresponding author: Hidetoshi Tanioka, Department of Oph-
thalmology, Kyoto Prefectural University of Medicine, 465 Kajii-cho,
Hirokoji-agaru, Kawaramachi-dori, Kamigyo-ku, Kyoto, 602-0841, Ja-
pan: htanioka@eye.ophth.kpu-m.ac.jp.
3820
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3/12 or secretory mucin2,5,6 and is necessary for ocular surface
homeostasis. In patients with severe ocular surface disorders
such as Stevens-Johnson syndrome (SJS), ocular cicatricial pem-
phigoid (OCP), and chemical injuries, the corneal epithelium
may be destroyed and replaced by conjunctival epithelium
(conjunctivalization). The ocular surface is often inflamed, vas-
cularized, opacified, and keratinized, and vision is severely
compromised.
Cultivated corneal stem cells712 and oral epithelia1315
transplantations are a newly developed surgical strategy in
which to treat such pathologic conditions. Although these
treatments were reported to be effective in applying regener-
ative medicine, several problems remain. For example, tissue
transplantation from allogeneic donors carries the risk of re-
jection and may require postoperative immunosuppressive
therapy that can induce severe systemic and local side effects.
The longevity of cultivated corneal and oral mucosal epithe-
lium remains to be investigated.
In addition to corneal and oral mucosal epithelium, con-
junctival epithelium is a third epithelial cell source that can be
cultivated to be transplanted for ocular surface reconstruction.
Among all stratified epithelial tissues in the body, these cells
are most akin biologically to corneal epithelial cells. Therefore,
conjunctival epithelial cells transplanted onto the corneal
surface may serve some of the functions of corneal epithelial
cells. As the transplantation of cultivated human conjuncti-
val epithelial cells (HCjE) succeeded in reconstructing the
conjunctiva of patients with various ocular surface conditions,
e.g., pterygium,16 20 we postulated that cultivated HCjE sheets
could be transplanted onto the corneal surface.
To test our hypothesis, we cultured HCjE on human amni-
otic membrane (AM) and transplanted them onto denuded
rabbit corneas. The transplanted HCjE were well-maintained
and remained clear and smooth during the postoperative pe-
riod. Histologic and immunohistochemical analyses revealed
that the engrafted epithelium shared the morphology and char-
acteristics of corneal epithelium, suggesting that cultivated
HCjE may represent a viable alternative to replace damaged
corneal epithelium.
METHODS
Human Subjects
This research was approved by the Committee for Ethical Issues on
Human Research of Kyoto Prefectural University of Medicine and
adhered to the tenets of the Declaration of Helsinki. Normal conjunc-
tival tissues were obtained from patients with conjunctivochalasis.
Human AM was harvested at the time of Cesarean section and pro-
cessed by previously reported methods.21 The procedures were care-
fully explained to all donors, and their prior informed consent for use
of their tissue was obtained.
Investigative Ophthalmology & Visual Science, September 2006, Vol. 47, No. 9
Copyright Association for Research in Vision and Ophthalmology
 IOVS, September 2006, Vol. 47, No. 9 Autologous Tissues Source for Corneal Transplantation 3821

Primary Culture of HCjE Cells

The cells were cultured according to a slightly modified, previously
reported system.22 Briefly, denuded human AM was placed on a porous
support membrane (Millipore Corp., Bedford, MA) with the epithelial
basement membrane side up. The membrane was then introduced into
wells of a six-well culture plate containing mitomycin-treated feeder
cells (NIH 3T3; American Type Culture Collection, Manassas, VA) to
achieve a dual-chamber culture. After a 1-hr incubation with 1.2 IU
dispase (Roche, Tokyo, Japan), the human conjunctival epithelium
(the area of this conjunctival source was 15 mm2) was removed from
the underlying stroma by mechanical scraping and further dissociated
by digestion with 0.1% Trypsin-EDTA. The HCjE cells were then seeded
on the upper chamber of the culture system and grown according to
a three-step culture regimen. Until they reached confluence (6 8
days), the cells were grown in low-calcium medium (Defined Keratin-
ocyte-SFM; Invitrogen, Tokyo, Japan) containing 2% FBS. After reach-
ing confluence, they were grown for 7 days in high-calcium medium
(mixture of Defined Keratinocyte-SFM and DMEM/F12/10% FBS at a
ratio of 1:1) to promote differentiation. They were then exposed to air
by decreasing the volume of the medium (air-lifting) over the course of
1 week to promote epithelial integrity. All cultures were incubated at
37C in a 5% CO2-95% air incubator. The medium was changed every
day or every other day.

Conjunctival Epithelium Transplantation onto

Rabbit Corneas
At all times, the rabbits were housed and treated in accordance with
the ARVO Statement for the Use of Animals in Ophthalmic and Vision
Research. All experimental procedures were approved by the Commit-
tee for Animal Research of Kyoto Prefectural University of Medicine.
Using eight Japanese white rabbits weighing 2.4 to 2.8 kg (OBS,
Kyoto, Japan), we performed superficial lamellar keratectomy to re-
move the entire corneal epithelium. To ensure complete removal of
the limbal epithelium, we surgically excised the entire limbal epithe-
lium and surrounding conjunctival tissue up to 2 mm from the limbus FIGURE 1. Cell culture of HCjE and transplantation into rabbit cornea.
from one eye, down to the bare sclera. The cultured HCjE sheets were
transplanted onto the denuded ocular surface to completely cover the
resected area and were sutured in place with 10-0 nylon (8 12 sutures
Immunostaining and Light-Microscopic Analysis
per sheet). The graft was then covered with a soft contact lens secured
with four peripheral anchoring sutures. Finally, tarsorrhaphy was per- Tissue sections (8 m) were placed on glass slides and subjected to
formed with 6-0 nylon sutures (Fig. 1B). After surgery, the rabbits were hematoxylin staining or indirect-immunostaining analysis. Briefly, the
treated with topical antibiotics (0.3% ofloxacin ointment; Santen Phar- sections were fixed with Zambonis fixative or acetone (4C, 5 min-
maceutical Co., Ltd, Osaka, Japan), triamcinolone acetonide (0.2 mL utes), immersed for 1 hour in blocking solution (1% BSA in 0.01M PBS),
injected subconjunctivally; Bristol-Myers Squibb Co., Tokyo, Japan), and treated with primary antibody solutions (Table 1) and normal
and systemic antibiotics (10 mg gentamicin/rabbit, delivered intramus- mouse IgG1, IgG2a, and IgG2b (Dako Cytomation Kyoto, Japan), and
cularly [IM]; Nacalai Tesque Inc. Kyoto, Japan). They also received a goat IgG (Santa Cruz Biotechnology Inc., Santa Cruz, CA) as the nega-
daily IM injection of 0.2 mg/kg of the immunosuppressant agent tive controls. After a 1-hour incubation, the sections were washed with
FK50623 (Astellas Co., Ltd., Tokyo, Japan) to inhibit a possible zeno- 0.01 M PBS and then treated with fluorescent secondary antibody
geneic reaction or nonspecific inflammation. solutions (Alexa-488-labeled anti-mouse IgG or anti-rabbit IgG; Invitro-
gen, Carlsbad, CA). After 1-hour incubation, the sections were washed
with 0.01 M PBS and mounted with medium containing an anti-
Slit Lamp Examination photobleaching reagent (3% Dabco; Wako Pure Chemical Industries
On the day of transplantation and on the 4th and 14th postoperative Ltd., Osaka, Japan). Fluorescent images of the sections were inspected
days, the ocular surface of the eight transplant recipients was exam- and photographed with a confocal laser scanning microscope (TCS-
ined and photographed with a slit lamp biomicroscope (SL-1600; SP2; Leica, Tokyo, Japan). Unless otherwise stated, all incubations
Nidek Co., Ltd., Aithi, Japan). were at room temperature.

Tissue Preparation Transmission Electron Microscopic Examination

Engrafted tissues were removed from the eyes of eight rabbits killed 14
days after transplantation. In vivo conjunctival tissues, cultivated HCjE
cells, and transplanted conjunctival tissues were divided into two
portions, one of which was embedded in optimal cutting temperature
compound (Tissue-Tek; Sakura Fine Technical Co., Ltd., Tokyo, Japan)
and snap frozen with liquid nitrogen for immunostaining analysis. The
other portion was processed for electron microscopy (EM).
Specimens were fixed in 2.5% glutaraldehyde in 0.1 M PB, washed 3
times in PB, and postfixed for 1 hour in 2% aqueous osmium tetroxide.
They were then passed through a graded ethanol series, transferred to
propylene oxide, and embedded in Epon-812 (TAAB, Berkshire,
England). Ultrathin sections were cut and stained with uranyl acetate
and lead citrate before examination under a TEM (H-7000; Hitachi,
Tokyo, Japan).

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 3822 Tanioka et al. IOVS, September 2006, Vol. 47, No. 9

TABLE 1. Antibodies Used in the Study

Type of Immunized
Group Antigen Dilution Antibody Animal Company* Annotation

Putative stem cell markers ABCG2 40 (Mo) M Kamiya ATP-binding cassette transporter
p63 100 (Mo) M Santa Cruz p53 homologous protein
Adhesion molecule Laminin5 100 (Mo) M Chemicon Hemidesmosome component protein
Integrin 6 100 (Mo) M Cymbus Hemidesmosome component protein
Integrin 4 100 (Mo) M Chemicon Hemidesmosome component protein
Desmoplakin 1 (Mo) M Progen Desmosome component protein
Nuclei Human nuclei 30 (Mo) M Chemicon Possible to distinguish human cells from
other animal cells
Cytokeratin CK3 50 (Mo) M Progen Major cytokeratin in corneal epithelium
CK4 100 (Mo) M Novocastra Major cytokeratin in nonkeratinizing
mucosal epithelium
CK12 100 (Po) G Santa Cruz Major cytokeratin in corneal epithelium
CK13 200 (Mo) M Novocastra Major cytokeratin in nonkeratinizing
mucosal epithelium
Mutin MUC4 50 (Mo) M Zymed A membrane-bound mucin
MUC5AC 100 (Mo) M Novocastra Secreted mucin/goblet cell mucin

Mo, monoclonal; Po, polyclonal; M, mouse; G, goat.

* Kamiya: Kamiya Biomedical Company, Seattle, WA; Santa Cruz: Santa Cruz Biotechnology Inc., Santa Cruz, CA; Chemicon: CHEMICON
International Inc., Temecula, CA; Symbus: Symbus Biotechnology LTD, Hampshire, UK; Progen: PROGEN Biotechnik GmbH, Heidelberg, Germany;
Novocastra: Novocastra Laboratories Ltd, Newcastle, UK; Zymed: ZYMED Laboratories Inc., South San Francisco, CA.

RESULTS
Analysis of HCjE Sheets
HCjE sheets, grown on AM for 3 weeks, manifested five to six
layers of well-stratified epithelium (Fig. 2A, 2D) without goblet
cells (Fig. 2C). Thus, they were similar to in vivo corneal
epithelium (Fig. 2B). The TEM examination revealed many
microvilli on the surface of the superficial cells (Fig. 2E),
desmosomes at intercellular junctions (Fig. 2F), and hemides-
mosomes on the basal side of the basal cells (Fig. 2G).

FIGURE 2. Histologic examination of HCjE cells grown on human amniotic membrane. Cultivated human conjunctival epithelium and in vivo
corneal- and conjunctival epithelium were examined by light microscopy (AC: semithin section stained with toluidine blue) or transmission
electron microscopy (DG). The cultivated epithelium was five to six layers thick (A, D) and exhibited typical microvilli (E), and desmosome- (F)
and hemidesmosome (G) formation.

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 IOVS, September 2006, Vol. 47, No. 9 Autologous Tissues Source for Corneal Transplantation 3823

FIGURE 3. Expression of putative markers for stem/progenitor cells and epithelial adhesion molecules in the cultivated HCjE. (A) In vivo human
limbal epithelium (Aa, Ab), in vivo HCjE (Ac, Ad), and cultivated HCjE (Ae, Af) were immunostained (green) with ABCG2 (Aa, Ac, Ae) or p63
(Ab, Ad, Af) and counterstained with propidium iodide (red). (B) In vivo HCjE (Ba, Bb, Bc, Bd) and cultivated HCjE (Be, Bf, Bg, Bh) were
immunostained (green) with laminin5 (Ba, Be), integrin 6 (Bb, Bf), integrin 4 (Bc, Bg), or desmoplakin (Bd, Bh) and counterstained with
propidium iodide (red).

Frozen sections of in vivo ocular tissues and cultivated HCjE
were subjected to indirect immunostaining analysis. The basal
cells of the cultivated HCjE sheets expressed the putative stem
cell markers ABCG2 and p63 (Fig. 3AaAf); their expression
patterns were almost identical with those of in vivo limbal
epithelium. The hemidesmosome component proteins laminin
5 and integrin 64 were restricted to the interface between
the basal cells and the AM. Desmoplakin, a desmosome-associ-
ated protein, was expressed at cell cell borders. These expres-
sion patterns were almost identical with those of in vivo HCjE
(Fig. 3BgBn).
Transplantation of Cultivated HCjE Sheets
Cultivated HCjE sheets were successfully transplanted onto the
cornea of all eight rabbits. The transplanted conjunctival epi-
thelium completely covered all corneas and remained transpar-
ent, smooth, and devoid of epithelial defects during the 2-week
postoperative observation period (Fig. 4). The transplanted
HCjE was well-maintained on the recipients corneal surface;
there were no instances of graft retraction or dislodgement.
The engrafted epithelium manifested five to six layers of strat-
ified squamous epithelium, rendering it morphologically simi-
lar to normal corneal epithelium (Figs. 5AD). We observed no
goblet cells in the engrafted epithelium. As the grafts stained
positive for the anti-human nuclei antibody that specifically
reacts with human tissue,24,25 we were able to confirm that the
epithelial cells on the rabbit corneas were of human origin
(Fig. 5E).
Histologic and EM Appearance of the Engrafted
Conjunctival Epithelium
The engrafted epithelium consisted of five to six well-stratified
layers harboring cuboidal or columnar basal cells, winged su-
prabasal cells, and flattened squamous superficial cells (Fig.
6A). There were many microvilli on the surface of the super-
ficial cells. Tight junction-like structures were present at the
cell cell border of the superficial cells (Fig. 6B), and desmo-
somes were at the intercellular regions of the epithelial cells
(Fig. 6C). Hemidesmosomes were seen at the basal cell-AM
substrate junction zone (Fig. 6D).

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 3824 Tanioka et al.
FIGURE 4. HCjE grafted onto the denuded rabbit cornea. The en-
grafted HCjE was inspected just after transplantation (A, B) and 4 (C,
D) and 14 days (E, F) after transplantation. The engrafted HCjE was
devoid of epithelial defects at all observation points.
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IOVS, September 2006, Vol. 47, No. 9
Immunohistochemistry
Although MUC4 and MUC5AC were expressed by HCjE in vivo
(Figs. 7A, 7D), neither cultivated nor engrafted HCjE cells
stained positive for MUC5AC (Figs. 7E, 7F). In vitro cultivated
HCjEs did not express MUC4, but engrafted HCjE was found to
express MUC4 (Figs. 7B, 7C). CK4/13, normally expressed in
conjunctival epithelium, was present in the cultivated HCjE
sheets (Figs. 7GL). In vivo conjunctival epithelium contained
a few CK3/12-positive cells, as did cultivated and engrafted
HCjE (Figs. 7MR).
DISCUSSION
We established a method for the culture of well-stratified con-
junctival epithelium on human AM. The epithelial sheets we
obtained exhibited high physical integrity, were well main-
tained after transplantation onto denuded rabbit corneas, and
contributed to corneal transparency. Our results suggest that it
may be possible to use these epithelial sheets for corneal
epithelial replacement in patients with various ocular surface
disorders.
It was initially intended in this study to culture rabbit con-
junctival epithelial cells for transplantation onto rabbit corneas
because this procedure is apparently free of undesirable xeno-
geneic rejection. However, the decision was made to trans-
plant the cultivated HCjE sheets onto rabbit corneas for the
following reasons. First, the optimal culture conditions for
rabbit and human cells are reportedly different.15,26,27 Consid-
FIGURE 5. Light microscopy of the
engrafted tissue and distribution of
human epithelial cells on the graft. At
2 weeks, the engrafted epithelium
demonstrated five to six layers of
stratified squamous epithelium simi-
lar to normal corneal epithelium (A
C). (D) Conjunctiva of the recipient.
No goblet cells were visible in the
grafted epithelium (B, C). The en-
grafted cornea (E) stained positively
with anti-human nuclei antibody
(green). Normal human cornea (F)
and rabbit cornea (G) served as pos-
itive and negative controls, respec-
tively. The nuclei were counter-
stained with propidium iodide (red).
() Suture track.
 IOVS, September 2006, Vol. 47, No. 9 Autologous Tissues Source for Corneal Transplantation
FIGURE 6. Transmission electron
microscopy of the engrafted tissue.
(A) Transmission electron micros-
copy at low magnification; (B) mi-
crovilli (arrowhead), tight junction
(arrow); (C) desmosome (arrow);
(D) hemidesmosome.
ering that our final goal is to translate our data to clinical
treatment, it is crucial to determine the optimal culture condi-
tion for making well-stratified HCjE epithelial sheets which
share sufficient physical integrity to tolerate intra- and postop-
erative surgical stress. Second, if cultivated rabbit conjunctival
epithelium is transplanted onto rabbit corneas, it is difficult to
discriminate between transplanted and migrated host-derived
cells. In contrast, a great advantage of this experimental system
was that the use of a specific antibody to human nuclei24,25
made it possible to identify which cells were of human origin.
To ensure complete removal of the limbal and corneal
epithelium, superficial lamellar keratectomy as well as com-
plete limbectomy down to bare sclera was performed. Al-
though we could not confirm that all the rabbit epithelial cells
were removed, the expression of human-specific antibodies in
the epithelial sheet helped to confirm that the epithelial cov-
ering was truly from the donor human tissue.
For the transplantation of HCjE to be successful, the culti-
vated sheet must possess structural integrity. The normal cor-
neal epithelium features desmosomes at the cell cell interface,
and their presence contributes to its structural integrity.
Hemidesmosomes at the basal cell surface serve to attach the
basal cells to the underlying basement membrane. We demon-
strated that desmosome-associated (desmoplakin),28 hemides-
mosome-associated (integrin 64),29 and basement mem-
brane-associated (laminin 5)30,31 proteins were present in the
cultivated HCjE sheets. Furthermore, as in the corneal limbus,
basal cells in the HCjE sheets expressed the putative stem cell
markers ABCG2 and p63,32,33 suggesting that they possess the
structural and regenerative characteristics of corneal and lim-
bal epithelium.34
MUC4, one of the mucin core proteins secreted from the
surface of in vivo conjunctival epithelium,6,35 was not ex-
pressed in the cultivated HCjE cells, although it was expressed
in the engrafted HCjE. In rats fed a retinoic acid depleted diet,
the expression of mucin genes by the ocular surface epithe-
lium was decreased.36 Therefore, it is possible that the culti-
vated HCjE failed to express MUC4 because the culture me-
dium lacked this solute factor. Alternatively, retinoic acid
present in rabbit tears may have led to the recovery of MUC4
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3825
expression in the engrafted HCjE. MUC5AC was not found to
be expressed in the goblet cells of conjunctival epithelium37,38
in either cultivated- or engrafted HCjE, although a series of
contiguous sections were inspected. Considering the previous
report that approximately 500 goblet cells exist in a 1-mm2
section of conjunctival epithelium,39 7500 goblet cells may
exist in the initial period of cultivation. However, no goblet
cells were identified, both in cultivated HCjE at the end stage
of the culture and engrafted HCjE at 2 weeks after surgery. This
suggests that our culture conditions did not support goblet cell
differentiation in culture or after transplantation.
We recently reported that similar to corneal epithelial cells,
as many as 1% of conjunctival epithelial cells are CK3/12
positive.40 We postulate that the CK3/12-positive cells in the
engrafted HCjE derived from the resected conjunctiva and
were maintained in our culture system. We documented else-
where41 that the expression of thrombospondin-1, an inhibitor
of vascularization, was much higher in corneal than conjunc-
tival epithelium. As the expression level of this gene by CK3/
12-positive cells in the engrafted HCjE was similar to the level
seen in corneal epithelium, it may contribute to the inhibition
of corneal neovascularization.
In patients with unilateral chemical or thermal injury, the
conventional repair by limbal autografts from the contralateral
eye requires 3 to 6 hours, and this may inflict iatrogenic limbal
stem cell deficiency on the donor eye. The transplantation of
autologous cultivated limbal stem cells has yielded promising
results and requires the harvest of much less tissue, thereby
reducing the risk of iatrogenic injury to the donor eye.8,42,43 To
treat bilateral ocular surface disorders such as SJS, our group
has reported allogeneic transplantation7 or more recently, au-
tologous cultivated oral epithelial transplantation, as promising
treatment options.14,15 We now add cultivated autologous con-
junctival epithelial transplantation for corneal epithelial re-
placement as a promising new modality to treat severe ocular
surface disorders. It may be safer than the conventional meth-
ods currently used, and immunologically, it is superior to
allogeneic transplantation. From a cytological point of view,
autologous conjunctival epithelium represents a better alterna-
tive than oral mucosal epithelium for corneal epithelial replace-
 3826 Tanioka et al.
ment. Because we were dealing with xenotransplantation, one
of the limitations of this study is the short follow-up period of
14 days. With more prolonged follow-up, it may be that some
conjunctival cells would differentiate into goblet cells and that
progressive conjunctivalization and neovascularization would
occur. More long-term studies are needed to investigate some
of these questions.
In summary, ours is the first report that clearly demon-
strates the potential of cultivated HCjE as an alternative tissue
source for replacement of the corneal epithelium. Our animal
study is a step toward the eventual transplantation of autolo-
gous cultivated HCjE to treat patients with ocular surface
disorders, and studies are ongoing to resolve outstanding is-
sues.
Acknowledgments
The authors thank John Bush and Christine Kime for reading the article
and the Northwest Lions EyeBank foundation for helping to obtain
fresh human corneal tissues.
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IOVS, September 2006, Vol. 47, No. 9
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